Effects of Prenatal Exposure to a Mixture of Organophosphate Flame Retardants on Placental Gene Expression and Serotonergic Innervation in the Fetal Rat Brain

There is a growing need to understand the potential neurotoxicity of organophosphate flame retardants (OPFRs) and plasticizers because use and, consequently, human exposure, is rapidly expanding. We have previously shown in rats that developmental exposure to the commercial flame retardant mixture Firemaster 550 (FM 550), which contains OPFRs, results in sex-specific behavioral effects, and identified the placenta as a potential target of toxicity. The placenta is a critical coordinator of fetal growth and neurodevelopment, and a source of neurotransmitters for the developing brain. We have shown in rats and humans that flame retardants accumulate in placental tissue, and induce functional changes, including altered neurotransmitter production. Here, we sought to establish if OPFRs (triphenyl phosphate and a mixture of isopropylated triarylphosphate isomers) alter placental function and fetal forebrain development, with disruption of tryptophan metabolism as a primary pathway of interest. Wistar rat dams were orally exposed to OPFRs (0, 500, 1000, or 2000 μg/day) or a serotonin (5-HT) agonist 5-methoxytryptamine for 14 days during gestation and placenta and fetal forebrain tissues collected for analysis by transcriptomics and metabolomics. Relative abundance of genes responsible for the transport and synthesis of placental 5-HT were disrupted, and multiple neuroactive metabolites in the 5-HT and kynurenine metabolic pathways were upregulated. In addition, 5-HTergic projections were significantly longer in the fetal forebrains of exposed males. These findings suggest that OPFRs have the potential to impact the 5-HTergic system in the fetal forebrain by disrupting placental tryptophan metabolism.

TGF-β1 EXPRESSION BY GLIOMA C6 CELLS IN VITRO

The aim of the work was to study the impact of fetal rat brain cell supernatant (FRBCS) on the expression of transforming growth factor β1 (TGF-β1) and p53 in C6 cells of rat glioma in vitro. Materials and Methods: FRBCS was obtained from suspensions of fetal rat brain cells on the 14th (E14) day of gestation. C6 glioma cells were cultured for 48 h in the presence of FRBCS or FRBCS + anti-TGF-β1 monoclonal antibody. Immunocytochemical staining for TGF-β1 and p53 was performed. Results: The proportion of TGF-β1-immunopositive tumor cells in C6 glioma cultures was statistically significantly higher than in the control cell cultures of normal fetal rat brain. FRBCS reduced the proportion of TGF-β1-immunopositive tumor cells and increased the proportion of p53-immunopositive cells in C6 glioma cultures. In cells cultured with FRBCS + anti-TGF-β1 monoclonal antibody, the above effects of FRBCS were abrogated. Conclusion: The obtained results suggest that TGF-β1 seems to be responsible for decrease in TGF-β1 expression and increase in p53 expression in C6 glioma cells treated with FRBCS.

Morphometric characteristics of TGF-β1-positive cells of fetal rat brain in vitro

One of the directions of cell therapy being developed for brain gliomas is the use of the neurogenic stem and progenitor cells (NSCs/NPCs). There are data on the anti-tumor and immunomodulating properties of the NSCs/NPCs the mechanisms of which were not disclosed yet. One of the potential targets for tumor therapy is the transforming growth factor β (TGF-β1) which is thought to be one of the key molecules in the regulation of proliferation, differentiation and cell survival or apoptosis. In the view of available information about the possibility of TGF-β1 production by the mammalian multipotent NSCs/NPCs, the aim of this work was to study the TGF-β1-positive cells in the dynamics of cultivation of fetal brain neurogenic cells as a potential source of anti-tumor or immunomodulating effects of these cells.Material and methods. The fetal rat brain cells on 14th (E14) day of gestation were used as the source for cultivation in standard conditions (DМЕМ + 1 % fetal bovine serum) and studied on the 2nd and 37thday by morphometry and immunocytochemistry.Results. In the fetal rat brain cell cultures, the TGF-β1-positive cells made 22.04 ± 2.33 % and the nestin-positive cells made 49.16 ± 10.60 % of the total cells number. The morphometric parameters of TGF-β1-positive cells exceeded the corresponding values of negative cells (average values of cross-sectional areas of the cytoplasm, cross-sectional areas of the nucleus, nuclear-cytoplasmic ratio). During cultivation the relative amount of TGF-β1-positive cells was slightly decreased 15.27 ± 9.80 % (p = 0.7) and their sizes were increased. On the 37th day of cultivation the sizes of TGF-β1-positive and their nuclei were smaller in the comparison with the TGF-β1-negative cells.Conclusions. The presence of TGF-β1 expression by part of neurogenic cells of fetal rat brain (E14) in vitro was found, which persisted throughout cultivation (~5 weeks). Significant quantitative differences of morphometric parameters of TGF-β1-positive and negative cells were detected.

Dynamics of CD133+ cells in cultures of glioma c6 and fetal rat brain under the neurogenic cells supernatant influence

Cellular and molecular similarities between brain tumor stem cells (BTSCs) and normal neurogenic stem cells (NSCs) motivate the search for new methods of treatment of malignant glioma using NSCs. CD133 molecule could be one of the most typical markers of BTSCs and considered as a target for therapy of brain tumors.The aim of this study was to evaluate the effect of rat neurogenic cells supernatant (NCsS) on the content of CD133+ cells in glioma C6 cell cultures.Materials and methods. The cells of rat brain glioma C6 were used as the source for the cultivation; for comparative assessment of tested compound impact on the intact nervous system the fetal rat brain cells on 14th (E14) day of gestation were used. The study was performed in control cultures under standard culture conditions without NCsS adding and tested cultures with adding NCsS (0.10 mg/ml of protein) for 48 hours. NCsS was received from suspensions of rat brain neurogenic cells (E14).Results. CD133-positive cells were 12.05 ± 4.77 % of the total number of cells in C6 glioma culture and 37.36 ± 12.33 % of the total number of cells in fetal rat brain culture. CD133-positive cells had a smaller size than negative cells (average values of cross-sectional area of cells and nucleus) and greater nuclear-cytoplasmic ratio. The cell and nucleus sizes of CD133-positive cells in cell cultures of fetal rat brain were twice larger than sizes of such cells in cultures of glioma C6.Under the conditions of NCsS for 48 hours the reducing in the number of CD133-positive cells in rat glioma C6 cell cultures (2.88 ± 0.41 %) and lack of such effects in cell cultures of fetal rat brain (E14) were found.Conclusion. The morphological differences of CD133-positive cells in glioma C6 cultures and in cell cultures of fetal rat brain (E14) were detected. The decrease of CD133-positive cells in glioma C6 cells culture under the influence of neurogenic cells supernatant was shown.